Approximately 40% of the total energy production of E. coli is consumed to synthesize the large number of ribosomal components: 3 RNAs (comprised of more than 400 nucleotides) and greater than 50 proteins. This observation and the direct correlation between the rates of E. coli growth and ribosome biogenesis indicate that an accurately assembled and functional ribosome is of utmost importance for cell viability. Our long term goal is to gain a detailed understanding of ribosome biogenesis and thus reveal the impact of this dynamic process on fundamental aspects of cell physiology, such as growth regulation. This proposal details studies of E. coli small (SOS) ribosomal subunit biogenesis. The purpose of this work is to gain an in-depth understanding of conformational changes and the factors that modulate these changes during the course of functional SOS subunit assembly. Three specific aims have been proposed to address these questions. First, conformational changes in 16S rRNA and the ribosomal proteins that facilitate these changes during different stages of assembly will be identified using a panel of chemical probing strategies. Second, using a combination of genetics and biochemistry, novel SOS subunit assembly intermediates formed in vivo will be identified and characterized. Two ribosomal protein mutant strains, including a chromosomal deletion of ribosomal protein S15, are in hand for this analysis. Third, extra- ribosomal factors that facilitate SOS subunit assembly will be analyzed. Factors identified in the current funding period will be explored in detail and new methods will lead to the identification and characterization of additional factors. Our progress during the current funding cycle and our preliminary data, both biochemical and genetic, indicate that all three specific aims will be successful and thus will directly impact our understanding of SOS subunit biogenesis. Such an understanding is critical for the identification of novel targets for attenuating bacterial growth and therefore for the development of antimicrobials.